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RECOMBINANT DNA

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These enzymes cleave double stranded DNA at specific sequences, ... Rare restriction nucleases , such as EcoRI, can be used for mapping of DNA. ... – PowerPoint PPT presentation

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Title: RECOMBINANT DNA


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RECOMBINANT DNA The ability to cut and paste
genetic material for further analysis is due
to the discovery of enzymes called restriction
endonucleases. These enzymes cleave double
stranded DNA at specific sequences, most
commonly formed by 4, 6 or 8 base pairs.
Different RE recognize sequences that are
either frequent or quite rare. They are useful
for different applications. Ex. EcoRI SmaI
3
More than 3000 REs have been isolated from
bacteria. Their function, in vivo, is to cleave
and promote degradation of viral DNA inside the
host bacterial cells. Bacterial DNA is protected
from cleavage because its methylated.
4
Restriction nucleases can cleave the DNA leaving
overhanging single stranded tails (sticky) or
blunt ends.
5
Rare restriction nucleases , such as EcoRI, can
be used for mapping of DNA . This analysis
involves cutting the sample DNA and separate the
fragments according to size by gel
electrophoresis.
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Agarose gel electrophoresis its prepared as a
1-2 agarose in buffer and cast horizontally on a
tray containing several wells, for each
sample. The bigger the DNA fragment the slower
it will migrate down the gel, when the current is
applied. Since the DNA is charged negatively, the
direction of the migration will be from the
negative to the positive electrode. The DNA is
visualized by addition of a dye (ethydium
bromide)that intercalates between the DNA bases
and is fluorescent. The intensity of fluorescence
is proportional to the amount of DNA in the
sample.
Show example
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Problem 2 agarose gel, in 50 ml TE
buffer Ingredients agarose powder
100x TE buffer
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DNA mapping Eco RI 1100 bp
500 bp 100 bp Bam H 900
800 Eco RI BamH 700
500
400 100
10
MOLECULAR CLONING The basic strategy in
molecular cloning is to insert a DNA fragment of
interest into a DNA molecule (called a vector)
that is capable of independent replication in a
host cell. The host cell is usually E. Coli, and
the vector is a plasmid or a phage that can
replicate producing million of progeny
recombinant molecules. Plasmid or phage DNA can
be isolated separately from the host genomic
DNA and identified by cutting and sequencing.
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Vectors for Recombinant DNA 1.Lambda (?) phage.
Its used for either genomic or cDNA clones from
eucaryotic cells.Sequences of DNA up to 15 kb
can be inserted. -Insertion -packaging -E.
Coli infection -Isolation of single clones
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2. Plasmids. Smaller than phage, easier to
manipulate, can replicate independently from the
host cell. Plasmid DNA can be easily separated
from the bacterial DNA and sequenced. 3. Cosmids
and yeast artificial chromosome (YAC) are used
To clone big pieces of genomic DNA (up to 45kb
in cosmid and over hundreds of kb in YAC)
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  • A plasmid vector is digested with EcoRI at a
    single site to produce two sticky ends.
  • A sample of human DNA is also digested with EcoRI
    to produce pieces with the same sticky ends.
  • Human DNA- or cDNA copied from mRNA using reverse
    transcriptase from retroviruses.
  • The two samples are mixed and allowed to
    hybridize, some molecules will form with pieces
    of human DNA inserted into the plasmid vector at
    the EcoRI site.
  • DNA ligase is used to covalently link the
    fragments.

Creating Recombinant DNA
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DNA denaturation the two strands are separated
by heat or chemical treatment. DNA/RNA
hybridization single stranded DNA or RNA is
allowed to anneal to its complementary strand
(either DNA or RNA) in controlled conditions
(temperature and salt concentration).
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How do we isolate large quantities of DNA for
further characterization? Polymerase chain
reaction (PCR)
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The techniques was developed by Nobel laureate
biochemist Kary Mullis in 1984 and is based on
the discovery of the biological activity at high
temperatures of DNA polymerases found in
thermophiles (bacteria that live in hot springs).
Most DNA polymerases work only at low
temperatures. But at low temperatures, DNA is
tightly coiled, so the polymerases don't stand
much of a chance of getting at most parts of the
molecules.
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But these thermophilic DNA polymerases work at
100C, a temperature at which DNA is denatured.
This thermophilic DNA polymerase is called Taq
polymerase, named after Thermus aquaticus, the
bacteria it is derived from. Taq polymerase,
however, has no proofreading ability. Other
thermally stable polymerases, such as Vent and
Pfu, have been discovered to both work for PCR
and to proofread.
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  • Weve got the sequence. Whats next.
  • Search databases to identify identical or similar
    sequences identified
  • by others and corresponding to known proteins.
  • You found a hit you can give your sequence a
    name
  • Your sequence is novel you characterize it

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An increasing number of resources is available on
the web to conduct searches. Ex. Sequence
characterization (amino acid translation,
presence of Intron/exon, promoter sequences,
structural analysis of polypeptides, Cellular
localization) Goal to get clues about the
identity or function of the candidate clone
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  • You found a putative peptide open frame sequence.
  • Q. Is it a real protein? How can I test it.
  • Gene expression in procaryotes. The isolated DNA
    is cloned in a
  • vector containing a T7 promoter. Add amino acids,
    ATP generating
  • system, T7RNA polymerase, E. Coli extract
    (containing ribosomes and
  • enzymes for translation).
  • Run the product of the reaction on a acrylamide
    gel to identify the
  • protein.

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Recombinant proteins can be also be expressed in
yeast or in mammalian cells. Applications
studies of protein function in particular tissues
or conditions (cancer).
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Polymerase Chain Reaction (PCR) It allows to
produce and isolate large amounts of single DNA
molecules for which the complete or partial
sequence is known. DNA Polymerase (Taq, Vent, or
Pfu) F and R oligonucleotides Free
deoxynucleotides Reaction buffer (includes
Mg) DNA Template (linear DNA, cDNA or genomic,
plasmid, pure, fixed, from cells, etc.) DNA is
amplified exponentially (1 copy 30 cycles 1
billion copies)
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PCR Variations RT-PCR Real time
PCR Degenerate primers PCR
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  • RT-PCR
  • Isolate RNA (total or polyA)
  • Convert to cDNA (complementary DNA, using the
    reverse transcriptase)
  • Use the DNA as template for the PCR reaction
  • Visualize fragment on agarose gel

27
Real time PCR Its used for accurate quantitation
of DNA samples. In real time PCR the
concentration of a DNA sample is proportional to
the amount of fluorescence generated at each
round of amplification.
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The real-time PCR system is based on the
detection and quantitation of a fluorescent
reporter. This signal increases in direct
proportion to the amount of PCR product in a
reaction. By recording the amount of
fluorescence emission at each cycle, it is
possible to monitor the PCR reaction during
exponential phase where the first significant
increase in the amount of PCR product correlates
to the initial amount of target template.
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The best method for quantitative detection of the
amplicon uses fluorescent probes. The TaqMan
probes use the fluorogenic 5' exonuclease
activity of Taq polymerase to measure the amount
of target sequences in cDNA samples. TaqMan
probes are oligonucleotides that contain a
fluorescent dye usually on the 5' base, and a
quenching dye on the 3' base. When irradiated,
the excited fluorescent dye transfers energy to
the nearby quenching dye molecule rather than
fluorescing (this is called FRET Förster or
fluorescence resonance energy transfer). Thus,
the close proximity of the reporter and quencher
prevents emission of any fluorescence while the
probe is intact.
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About People DNA Sequencing Oligo Synthesis
Microarray Real-Time PCR ABI-Freezer
Program Seqweb
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  • Degenerate PCR Degenerate Primers - What are
    they? Primers which have a number of options at
    several positions in the sequence to allow
    annealing to and amplification of a variety of
    related sequences. eg 5-TCG AAT TCI CCY AAY
    TGR CCN T-3 Y pYrimidines C / T (degeneracy
    2X) R puRines A / G (degeneracy 2X) I 
    Inosine  C / G / A / T  N Nucleotide C /
    G / A / T (degeneracy 4X)  
  • Why... use degenerate primers?
  • to amplify (fish out) conserved sequences of a
    gene or genes from the genome of an organism.
  • to get the nucleotide sequence after having
    sequenced some amino acids from a protein of
    interest

32
Detection of nucleic acids Based on the
principle of nucleic acid hybridization Southern
blot (DNA) Northern blot (RNA) In situ
hybridization (intact chromosomes, cells, tissue
slices, or embryos).
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Southern Blot
A DNA probe is hybridized to genomic DNA or
cDNA. The DNA probe is labeled (radioactive,
fluorescent, or chemoluminiscent). Southern
blotting was named after Edward M. Southern who
developed this procedure at Edinburgh University
in the 1970s. DNA molecules are transferred from
an agarose gel onto a membrane. Southern blotting
is designed to locate a particular sequence of
DNA within a complex mixture. For example,
Southern Blotting could be used to locate a
particular gene within an entire genome.
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  • Digest the DNA with an appropriate restriction
    enzyme.
  • 2. Run the digest on an agarose gel.
  • 3. Denature the DNA (usually while it is still on
    the gel).For example, soak it in about 0.5M
    NaOH, which would separate  double-stranded DNA
    into single-stranded DNA. Only ssDNA can
    transfer.                                      
                                  

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5. Probe the membrane with labeled ssDNA. This
is also known as hybridization.Whatever you call
it, this process relies on the ssDNA hybridizing
(annealing) to the DNA on the membrane due to the
binding of complementary strands. Probing is
often done with 32P labeled ATP,
biotin/streptavidin or a bioluminescent probe.
37
6. Visualize your radioactively labeled target
sequence. If you used a radiolabeled 32P probe,
then you would visualize by autoradiograph.
Biotin/streptavidin detection is done by
colorimetric methods, and bioluminescent
visualization uses luminescence.  
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Northern Blot Total RNA or poly(A) RNA is
isolated on agarose gel, transferred to a nylon
membrane and hybridized to a DNA probe. Usually,
the labeled DNA probe is a region of a gene for
which you want to study the expression
pattern. Example Is the estrogen receptor
expressed in all tissues at the same level?
40
In situ hybridization The probe is hybridized to
either RNA or DNA on a slice of tissue, or cells
in culture. The fluorescent microscope is used to
visualize the bound probe. Example in which
tissue of the developing embryo is protein X
first expressed?
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Detection of proteins Antibodies (polyclonal and
monoclonal) Western blotting - protein extract
- primary antibody
(mouse or rabbit) -
secondary antibody (labeled, goat IgG anti mouse
or rabbit)
-autoradiography
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Gene function Gene transfer in plants and
animals. DNA transfection transient or
stable Methods calcium phosphate precipitation
liposomes
electroporation
retrovirus Transgenic animals
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  • Microinjection of DNA into a pro-nucleus
  • of a fertilized mouse egg.
  • Eggs are transfected to foster mothers and are
    allowed to develop
  • Some of the offspring will have the injected DNA
    incorporated
  • in their genome.
  • 2. Embryonal stem cells are derived from
    blastocysts.
  • Foreign DNA is transfected into ES cells.
    Transformed cells
  • are injected into blastocysts, which are
    transferred to foster
  • mothers. Chimera offspring is produced, mated to
    normal male,
  • transgene is incorporated in the offspring.

44
Plant viruses and plasmids Ti plasmid (from the
Agrobacterium tumifaciens). The bacterium
attaches to the leaves of plants and the Ti is
transferred into plant cells where it becomes
incorporated into chromosomal DNA. So vectors
developed from Ti plasmids can be used as means
of introducing recombinant DNA into sensitive
plant cells.
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In vitro mutagenesis Single point
mutations Deletions Duplications Insertions
Reverse genetics
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Homologous recombination of a DNA molecule with
its chromosomal copy. A mutated DNA can be
integrated into the genome. The target gene can
be inactivated, so the function can be
identified. Antisense nucleic acids (RNA or
single stranded DNA) Dominant inhibitory
mutants. Mutant proteins can be introduced into
cells by gene transfer and used to study the
effects of blocking normal gene function
(embryonic development in xenopus)
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